Filamentous actin destabilization by H2O2 favors DnmA aggregation, with crucial roles of cysteines 450 and 776 in mitochondrial and peroxisomal division in Aspergillus nidulans.

IMPORTANCE: Mitochondria constitute major sources of H2O2 and other reactive oxygen species in eukaryotic cells. The division of these organelles is crucial for multiple processes in cell biology and relies on highly regulated mechano-GTPases that are oligomerization dependent and belong to the dynamin-related protein family, like A. nidulans DnmA. Our previous work demonstrated that H2O2 induces mitochondrial constriction, division, and remodeling of the outer membrane. Here, we show that H2O2 also induces a DnmA aggregation consistent with higher-order oligomerization and its recruitment to mitochondria. The study of this response uncovered that H2O2 induces the depolymerization and reorganization of actin as well as the critical role that cysteines 450 and 776 play in DnmA function. Our results provide new insights into the mechanisms of reactive oxygen species cell signaling and how they can regulate the dynamics of the actin cytoskeleton and the division of mitochondria and peroxisomes.

H2O2 Induces Calcium and ERMES Complex-Dependent Mitochondrial Constriction and Division as Well as Mitochondrial Outer Membrane Remodeling in Aspergillus nidulans.

The dynamin-like protein DnmA and its receptor FisA are essential for H2O2-induced mitochondrial division in Aspergillus nidulans. Here, we show that in the absence of DnmA or FisA, mitochondria show few spontaneous transient constrictions, the frequency of which is extensively increased by H2O2 or the carbonyl cyanide m-chlorophenyl hydrazone (CCCP). While H2O2-induced constrictions are transient, CCCP induces a drastic and irreversible alteration of mitochondrial filaments. H2O2 induces a gradual mitochondrial depolarization, while CCCP-induced depolarization is abrupt. The calcium chelator BAPTA-AM prevents the formation of mitochondrial constrictions induced by either H2O2 or CCCP. H2O2 also induces major rearrangements of the mitochondrial outer membrane, which remain after constrictions dissipate, as well as changes in endoplasmic reticulum (ER) and nuclear morphology. Similar mitochondrial constriction, ER and nuclear morphology changes are detected during the early stages of asexual development. ER and ER-Mitochondria encounter structure (ERMES) complex-composed of proteins Mdm10, Mmm1, Mdm43 and Mdm12-are important for mitochondrial division in Saccharomyces cerevisiae. As the Mdm10 ortholog MdmB was found to be essential in A. nidulans, we evaluated its functions in ΔmdmB terminal mutants and ΔmdmB heterokaryons. ΔmdmB conidia produce a short germ tube that fails to grow further, in which inherited mitochondria become gigantic and round shaped, lacking clear contacts with the ER. In slow-growing ΔmdmB heterokaryotic mycelia, multiple hyphae contain very long mitochondria with high ROS levels, as occur in ΔdnmA and ΔfisA mutants. In this hyphae, H2O2 fails to induce mitochondrial constrictions but not outer mitochondrial membrane reshaping, indicating that these are two separate effects of H2O2. Our results indicate that H2O2 induces a generalized mitochondrial constriction response, prior to actual division, involving gradual depolarization; they also indicate that Ca2+ and the ERMES complex are critical for both mitochondrial constriction and division. This supports a view of mitochondrial dynamics as the result of a cascade of signaling events that can be initiated in vivo by H2O2.

On the use of n-octyl gallate and salicylhydroxamic acid to study the alternative oxidase role.

The alternative oxidase (AOX) catalyzes the transfer of electrons from ubiquinol to oxygen without the translocation of protons across the inner mitochondrial membrane. This enzyme has been proposed to participate in the regulation of cell growth, sporulation, yeast-mycelium transition, resistance to reactive oxygen species, infection, and production of secondary metabolites. Two approaches have been used to evaluate AOX function: incubation of cells for long periods of time with AOX inhibitors or deletion of AOX gene. However, AOX inhibitors might have different targets. To test non-specific effects of n-octyl gallate (nOg) and salicylhydroxamic acid (SHAM) on fungal physiology we measured the growth and respiratory capacity of two fungal strains lacking (Ustilago maydis-Δaox and Saccharomyces cerevisiae) and three species containing the AOX gene (U. maydis WT, Debaryomyces hansenii, and Aspergillus nidulans). For U. maydis, a strong inhibition of growth and respiratory capacity by SHAM was observed, regardless of the presence of AOX. Similarly, A. nidulans mycelial growth was inhibited by low concentrations of nOg independently of AOX expression. In contrast, these inhibitors had no effect or had a minor effect on S. cerevisiae and D. hansenii growth. These results show that nOg and SHAM have AOX independent effects which vary in different microorganisms, indicating that studies based on long-term incubation of cells with these inhibitors should be considered as inconclusive.

DnmA and FisA Mediate Mitochondria and Peroxisome Fission, and Regulate Mitochondrial Function, ROS Production and Development in Aspergillus nidulans.

The dynamin-like protein Drp1 and its receptor Fis-1 are required for mitochondria and peroxisome fission in animal and yeast cells. Here, we show that in the fungus Aspergillus nidulans the lack of Drp1 and Fis-1 homologs DnmA and FisA has strong developmental defects, leading to a notable decrease in hyphal growth and asexual and sexual sporulation, with some of these defects being aggravated or partially remediated by different carbon sources. Although both DnmA and FisA, are essential for mitochondrial fission, participate in peroxisomal division and are fully required for H2O2-induced mitochondrial division, they also appear to play differential functions. Despite their lack of mitochondrial division, ΔdnmA and ΔfisA mutants segregate mitochondria to conidiogenic cells and produce viable conidia that inherit a single mitochondrion. During sexual differentiation, ΔdnmA and ΔfisA mutants develop fruiting bodies (cleistothecia) that differentiate excessive ascogenous tissue and a reduced number of viable ascospores. ΔdnmA and ΔfisA mutants show decreased respiration and notably high levels of mitochondrial reactive oxygen species (ROS), which likely correspond to superoxide. Regardless of this, ΔdnmA mutants can respond to an external H2O2 challenge by re-localizing the MAP kinase-activated protein kinase (MAPKAP) SrkA from the cytoplasm to the nuclei. Our results show that ROS levels regulate mitochondrial dynamics while a lack of mitochondrial fission results in lower respiration, increased mitochondrial ROS and developmental defects, indicating that ROS, mitochondrial division and development are critically interrelated processes.

SakA and MpkC Stress MAPKs Show Opposite and Common Functions During Stress Responses and Development in Aspergillus nidulans.

Stress activated MAP kinases (SAPKs) of the Hog1/Sty1/p38 family are specialized in transducing stress signals. In contrast to what is seen in animal cells, very few fungal species contain more than one SAPK. Aspergillus nidulans and other Aspergilli contain two SAPKs called SakA/HogA and MpkC. We have shown that SakA is essential for conidia to maintain their viability and to survive high H2O2 concentrations. H2O2 induces SakA nuclear accumulation and its interaction with transcription factor AtfA. Although SakA and MpkC show physical interaction, little is known about MpkC functions. Here we show that ΔmpkC mutants are not sensitive to oxidative stress but in fact MpkC inactivation partially restores the oxidative stress resistance of ΔsakA mutants. ΔmpkC mutants display about twofold increase in the production of fully viable conidia. The inactivation of the SakA upstream MAPKK PbsB or the simultaneous elimination of sakA and mpkC result in virtually identical phenotypes, including decreased radial growth, a drastic reduction of conidiation and a sharp, progressive loss of conidial viability. SakA and to a minor extent MpkC also regulate cell-wall integrity. Given the roles of MpkC in conidiation and oxidative stress sensitivity, we used a functional MpkC::GFP fusion to determine MpkC nuclear localization as an in vivo indicator of MpkC activation during asexual development and stress. MpkC is mostly localized in the cytoplasm of intact conidia, accumulates in nuclei during the first 2 h of germination and then becomes progressively excluded from nuclei in growing hyphae. In the conidiophore, MpkC nuclear accumulation increases in vesicles, metulae and phialides and decreases in older conidia. Oxidative and osmotic stresses induce MpkC nuclear accumulation in both germinating conidia and hyphae. In all these cases, MpkC nuclear accumulation is largely dependent on the MAPKK PbsB. Our results indicate that SakA and MpkC play major, distinct and sometimes opposing roles in conidiation and conidiospore physiology, as well as common roles in response to stress. We propose that two SAPKs are necessary to delay (MpkC) or fully stop (SakA) mitosis during conidiogenesis and the terminal differentiation of conidia, in the highly prolific phialoconidiation process characteristic of the Aspergilli.

Effect of textile dyes on activity and differential regulation of laccase genes from Pleurotus ostreatus grown in submerged fermentation.

This research was conducted to extend the knowledge on the differential regulation of laccase genes in response to dyes. In order to accomplish this, we analyzed both, the expression of five laccase genes by real time RT-qPCR, and also the laccase activity and isoforms patterns during the time-course of a Pleurotus ostreatus submerged fermentation supplemented with either acetyl yellow G (AYG) or remazol brilliant blue R (RBBR) dyes. For the purpose of obtaining a stable reference gene for optimal normalization of RT-quantitative PCR gene expression assays, we tested four candidate reference genes. As a result of this analysis, gpd was selected as reference index for data normalization. The addition of dyes had an induction effect on the enzymatic activity and also modified the zymogram profile. Fermentation with RBBR showed the highest laccase activity and number of isoforms along the course of the fermentation. Laccase gene expression profiles displayed up/down regulation along the fermentation time in four laccase genes (pox4, pox3, poxa1b and pox2), while pox1 was not expressed in either of the fermentation conditions. AYG addition caused the highest induction and repression levels for genes pox3 and poxa1b respectively. The expression level for all genes in the presence of RBBR were lower than in AYG, being in both conditions this response growth time dependent. These results show the influence of the nature of dyes on the induction level of laccase activity and on the differential regulation of the laccase genes expression in P. ostreatus.